The ability of microbial pathogens to overcome the normally highly polarized host mucosal epithelial barrier is an early and critical step in pathogenesis. This is particularly critical for opportunistic pathogens such as Pseudomonas aeruginosa (PA), one of the most virulent opportunistic pathogens of man. In the setting of epithelial injury and loss of cell polarity PA can effectively colonize the mucosal surfaces, cause further damage, and disseminate. We have recently discovered that binding of PA to the apical (AP) surface of cultured epithelial cells induces a cell reprogramming process in which the local plasma membrane transforms from apical (AP) to basolateral (BL) membrane, generating a microenvironment that facilitates bacterial colonization and entry into epithelial cells. We have demonstrated through a large body of evidence that activation of Phosphoinositide 3-kinase (PI3K) is a key step in this process. A very important question that has not been addressed is what is the signaling pathway that is activated upon binding that results in bacterial uptake and creation of this new microenvironment. PI3Ks are adapted to receive regulatory input from protein tyrosine kinases or tyrosine phosphorylated proteins. It is known that PA infection of epithelial cells induces changes in the tyrosine phosphorylation status of host proteins. We hypothesize that PA infection of epithelial cells causes a tyrosine phosphorylation signaling cascade leading to activation of PI3K. Our long-term goal is to understand how PA interacts with host cells and causes disease. Our short-term goal is to dissect the signaling pathway that PA utilizes to transform AP into BL membrane during AP entry into epithelial cells. We outline two specific aims to dissect the signaling pathway(s) between binding of PA and activation of PI3K. In aim 1, we take a candidate protein approach and explore the role of specific host receptor and non-receptor tyrosine kinases in PA-mediated activation of PI3K and resultant changes in the AP membrane. Many of these candidate genes were identified in the RNAi screen that was carried out in the parent grant. In aim 2, we outline two complementary approaches to identify host proteins whose tyrosine phosphorylation changes in response to PA binding to the AP surface of polarized MDCK cells. These studies will provide a mechanistic insight into the process of internalization triggered by PA after attachment to the host cell epithelium. They will identify host factors that the bacteria exploit to cause disease. The elucidation of PA-host cell interactions at the molecular level will open the door for the development of new drugs that target not the pathogenic bacteria itself but the key mechanisms that it uses to invade and cause disease. This research will be done primarily in Uruguay at Institut Pasteur de Montevideo in collaboration with Arlinet Kierbel, as an extension of NIH Grant No. R01AI065902. 7. Public Health Relevance: Pseudomonas aeruginosa is one of the most virulent opportunistic pathogens of man and is the causative agent of a variety of acute and chronic infections. We are investigating how this bacterium infects epithelial cells by subverting the host cell machinery. New identify host cell proteins involved in Pseudomonas infection can potentially be used as novel drug targets.